PROJECT SUMMARY/ABSTRACT Triple-negative breast cancer (TNBC) is an aggressive disease that grows quickly and metastasizes early. There are no effective therapies for metastatic TNBC, so new strategies are needed. We will meet this need using nanoparticles (NPs) we developed that interfere with Wnt signaling, the key driving force behind TNBC growth and metastasis. The main effector of Wnt signaling is ?-catenin, and it is activated in TNBC cells when wnt ligands bind frizzled7 (FZD7) receptors that are overexpressed on TNBC cells relative to normal cells. Active ?-catenin promotes TNBC growth and metastasis by amplifying the expression of genes that support cell survival, proliferation, stem-like behavior, migration, and invasion. Our central hypothesis is that suppressing Wnt signaling at two levels will reduce TNBC cells? metastatic potential, so we have created Wnt inhibitory NPs coated with both FZD7 antibodies and ?-catenin small interfering RNAs (siRNAs) to accomplish this task. The FZD7 antibodies enable TNBC cell binding and prevent Wnt signaling from being activated by blocking wnt ligands from binding FZD7. The siRNAs further suppress Wnt signaling by directly inhibiting ?- catenin through RNA interference. Our preliminary studies indicate that these NPs can enter existing TNBC lung metastases in mouse models to inhibit their growth. In the proposed work we will elucidate the mechanism of action of these NPs in greater detail by studying their interaction with TNBC cells in vitro (Aim 1), validate that they can penetrate existing metastases in mice to halt their growth (Aim 2), and show that they can safely prevent the formation of new metastases in immune competent tumor-bearing mice (Aim 3). All aims will compare NPs that co-deliver FZD7 antibodies and ?-catenin siRNAs to NPs that deliver either agent individually in order to reveal if their combined effects are additive or synergistic. In addition to studying the NPs? impact on TNBC cells, we will also evaluate their effect on non-cancerous cells from breast, lung, and bone tissue in order to confirm they have negligible off-target effects. We will further study their safety in Aim 3 by monitoring animal weight, blood chemistry, and major organ histopathology. We expect to find that these NPs can inhibit Wnt signaling in TNBC cells to reduce their metastatic potential without impacting non- cancerous cells, enabling them to be used as tools to either treat existing metastases or prevent formation of new metastases. We also expect for NPs that co-deliver FZD7 antibodies and ?-catenin siRNAs to be more effective than NPs that deliver either agent individually. In summary, this project will evaluate Wnt inhibitory NPs as tools to treat and/or prevent TNBC metastasis. Unlike current therapies, which have unpredictable and insufficient results, our NPs will directly target TNBC cells and reduce their ability to grow and metastasize by suppressing Wnt signaling. If successful, this therapy will improve survival and quality of life for patients with metastatic TNBC, and it may ultimately be expanded to other cancers with different molecular drivers by altering the antibodies and siRNAs that are delivered with the NPs.